Diabetes is a chronic disease characterised by the onset of hyperglycaemia. This metabolic disorder arises either upon failure of the pancreas to secrete effective concentrations of insulin in the case of type 1 diabetes, or as a result of a build up of resistance by cells towards any bio-available insulin (type 2). Insulin is a peptidic hormone required by the body to transport glucose from the bloodstream to cells for energy. Failure of its production or effective use results in impaired glucose metabolism, and thus allows abnormally high quantities of this sugar to accumulate in the blood. In 2003, international surveys showed 194 million people have diabetes, and it is widely considered to be the world's fastest growing disease. Over one million Australians suffer from this disorder and it is Australia's sixth leading cause of death. These figures are cause for concern and unfortunately at present there is no cure. Diabetes is however manageable, but treatments such as insulin injections must be carried out everyday over a patient's lifetime after diagnosis, to allow a healthy and fulfilling life.
Discovery and isolation of the insulin molecule by Fredrick Banting and Charles Best in 1921 provided a significant breakthrough for those suffering diabetes, whose treatments until this time included only starvation diets. Medical benefits aside, scientists found insulin to be a fascinating molecule. It was the first protein to have its primary structure elucidated, and was the focal point of many Nobel Prize winning research projects. Knowledge of the key molecular determinants of insulin function is important not only for examining the downstream pathways leading to its physiological effects but also the development of new clinical compounds and biochemical/pharmacological probes. Despite the fact that nearly 85 years have passed since insulin was first isolated and 35 years since its structure was determined by X-ray crystallography, a high resolution, three dimensional structure of the insulin-receptor complex is still unavailable. Consequently, several key aspects of insulin's biochemistry remain to be discovered and understood. Recent studies using synthetic insulin analogues support the concept that insulin binds asymmetrically to two discrete sites within the receptor dimer, however, the biologically active conformation of the insulin molecule, complete structural features that constitute the receptor-binding domain, and the mechanism of insulin receptor activation, remain unknown.
The insulin receptor is a tetrameric integral membrane glycoprotein consisting of two 735 amino acid α-chains and two 620 amino acid β-chains. The α-chain resides on the extracellular side of the plasma membrane and contains the cysteine-rich insulin binding domain. Covalent insulin-receptor complexes have been isolated, however, the exact nature of this molecular interaction has yet to be fully elucidated. Insulin is firstly synthesised as a pre-prohormone precursor containing signal peptide-B-C-A domains. Mature insulin, however, consists of a 51 residue A-B heterodimer that is covalently linked by two interchain disulfide bonds. An intrachain disulfide bridge is also located within the insulin A-chain. The circulating and biologically active form of insulin is monomeric and its primary structure has been determined for at least 100 vertebrate species (FIG. 1). Of these, only six cysteine residues and ten other amino acids are fully conserved during evolution. The primary structure of insulin from several species is shown in FIG. 1. Invariance of these residues may be indicative of their key roles in receptor binding and/or maintaining a biologically active conformation.
Disulfide bonds serve structural and functional roles in peptides. In some peptides, the disulfide is involved in disulfide exchange chemistry at the receptor resulting in activation of receptors and downstream signaling. Yet in other peptides, the S—S motif serves only to preserve and/or create a bioactive conformation of the peptide. In such cases, chemistry can be used to replace the native disulfide with other bridging amino acid residues. Structural alteration will be well tolerated if key receptor interactions can be preserved and this will depend on the surrogate motif's ability to replicate native peptide tertiary and secondary structure. The role of each of insulin's disulfide bridges is currently unknown but it is highly likely that they play a key role in regulating insulin's function at its cell surface tyrosine kinase receptors. Invariance of the cysteine residues across the species highlights the importance of the S—S bridges to insulin's structure and function. The cystine framework found in insulin is also found in other so called ‘insulin superfamily’ molecules, e.g. relaxin. Despite their framework similarity to insulin, however, the relaxins bind and activate a different receptor, the G-protein coupled receptor, and are responsible for remarkably distinctive biological roles. Hence, nature has capitalized on a generic disulfide template to perform diverse neuroendocrine through to homeostasis roles.
Problems with insulin as a drug still exist and improved analogues are continually being sought. For example, insulin can only be taken by injection, as oral delivery results in protein cleavage by digestive enzymes before it can be absorbed into the bloodstream. In addition, insulin is generally refrigerated before use so that it does not degrade before it is injected. There is therefore a need for insulin analogues which display enhanced stability to proteolyic enzymes and those which can be stored at room temperature to facilitate transport and simplify storage.
Several studies have been conducted to determine the role of disulfide bonds in insulin and all have concluded that each cystine bridge, to varying degrees, is required to maintain biological function. In previous studies, however, cysteine residues have been replaced by non-bridging amino acids such as alanine and serine via site-directed mutagenesis. Previous research is difficult to analyse, since loss of biological activity cannot be unambiguously attributed to either unfolded protein structure and hence loss of a binding domain, or loss of a reactive disulfide motif. There is therefore a need to further investigate the role of the disulfide bonds in insulin.
Although insulin therapy provides a better quality of life for those afflicted with diabetes, it also remains a difficult drug to use. A narrow therapeutic index, inconsistent magnitude of effect, poor oral availability, weight gain, poor stability and limited potency are just of some of insulin's many limitations. Each of these make it very difficult for a diabetic patient to treat their disease and maintain the tightly regulated glucose concentrations of a healthy individual. The achievement of a “normal” 24 hour physiological insulin profile is nearly impossible to mimic. The nature of meals, exercise regimes, sleeping patterns, development of infections and endogenous production of glucose of the liver, are just some of the many causes of fluctuation in blood glucose levels. It is essential that basal insulin levels are maintained in the body 24 hours a day, but additionally extra supplies (bolus) are needed for the management of glucose ingested at meal times. Unfortunately, however, the ideal therapeutic window for insulin dosing is very narrow (4-6 mmol), and one of the major drawbacks of intensive insulin treatments is the potential for hyperglycaemic events (low blood sugar levels). The advent of recombinant access to insulin in the early 1980s, led to the development of analogues that are specifically engineered to accomplish slower, faster and more predictable activity profiles that help with timing, dosing and the maintenance of more stable physiological insulin levels (FIGS. 2A and 2B). Existing insulin analogues still, however, suffer from storage and stability problems.
Although there are a number of commerically available analogues of insulin, there is still a considerable desire to improve basal insulin treatments. This is partly because some of these existing analgoues have significant drawbacks. For example, some show enhanced IGF-1 (insulin-like growth factor 1) receptor binding activity which can result in enhanced mitogenic potency. Other analogues possess reduced potency in vivo. Accordingly, there is a need for insulin analogues with superior therapeutic profiles, as well as analogues which are able to retain all of the beneficial properties of the unmodified native insulin sequence while addressing the inherent physicochemical instability.